calcimycin and Cataract

Elevation of lens calcium occurs in both human and experimental animal cataracts, and opacification may result from calcium-activated proteolysis. The purpose of the present study was to determine whether calcium accumulation in cultured human and Macaca mulatta lenses results in proteolysis of crystallins, the major lens proteins.. Two-dimensional electrophoresis and mass spectrometry were used to construct detailed maps of human and monkey lens crystallins so that proteolysis after calcium accumulation could be monitored and the altered crystallins identified. Human and macaque lenses cultured in A23187 showed elevated lenticular calcium and superficial cortical opacities. The carboxypeptidase E (CPE) gene is expressed in human lens, and its presence in lens fibers was demonstrated by Western blot. To investigate whether CPE could cause similar truncation, purified alphaB-crystallin and CPE were incubated in vitro.. The major change observed in the crystallins of these cultured lenses was the accumulation of alphaB(1-174)-crystallin resulting from the loss of a C-terminal lysine. This result was significant, because similar appearance of alphaB(1-174) is a prominent change in some human cataracts. alphaB-crystallin and CPE incubation result in the formation of alphaB(1-174)-crystallin. This truncation was specific to alphaB(1-174)-crystallin, since other crystallins were not proteolyzed. Although a weaker activator than zinc, calcium activated CPE in vitro.. Since zinc concentrations did not increase during culture in A23187, calcium uptake in the lens may be responsible for CPE activation and alphaB(1-174) formation during cataract.

Topics: Aged; Aged, 80 and over; alpha-Crystallin B Chain; Animals; Blotting, Western; Calcimycin; Calcium; Carboxypeptidase H; Cataract; Child, Preschool; Electrophoresis, Gel, Two-Dimensional; Electrophoresis, Polyacrylamide Gel; Gene Expression Regulation, Enzymologic; Humans; Ionophores; Lens, Crystalline; Lysine; Macaca mulatta; Mass Spectrometry; Organ Culture Techniques; Zinc

Cataract is a multifactorial disease, and a large variety of stressors induce cataracts. Many cataractogenic stressors and endoplasmic reticulum (ER) stressors induce the unfolded protein response (UPR) in various cell types. The UPR is known to produce reactive oxygen species (ROS) prior to the inducement of apoptosis. We investigated whether ER stressors induce the UPR in lens epithelial cells (LECs) or whole rat lenses. Our results showed that higher levels of ER stressors activated Bip/GRP78, ATF4, and caspase-12. In addition, ROS were produced, free glutathione was decreased, and apoptosis was induced. LECs in the mitotic zone were the most susceptible to the UPR while the central LECs were the most resistant. The UPR induced the production of ROS in the ER and probably in the mitochondria. The detectable ROS production in cultured lenses is limited to the epithelial cells. These findings indicate that ER stressors induce the UPR in LECs with and without the induction of apoptosis, and we conclude that the UPR is probably one of the initiating factors of many types of cataracts.

Topics: Animals; Calcimycin; Cataract; Cell Line, Transformed; Endoplasmic Reticulum Chaperone BiP; Epithelial Cells; Glucose; Glutathione; Glycosylation; Homocysteine; Humans; Ionophores; Lens, Crystalline; Molecular Chaperones; Protein Folding; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Tissue Culture Techniques; Tunicamycin

The human genome contains 14 genes for 80 kDa catalytic subunit of the calcium-activated protease calpain (EC 34.22.17), yet no calpain-like cleavage sites have been detected on human lens crystallins in vivo. The purpose of the present study was to provide a comprehensive study of calpain activation in human and macaque lenses developing experimental cataract due to lens culture in ionophore A23187. Zymography was used to measure calpain activity; SDS-PAGE and immunoblotting were used to detect hydrolysis of potential lens protein substrates. Quantitative PCR was used to measure transcripts for calpains and the endogenous inhibitor calpastatin. We found that the lack of appreciable calpain-induced proteolysis in primate lenses is most likely due to relatively low levels of endogenous calpain activity compared to the high levels of endogenous calpain inhibitor, calpastatin.

Topics: Aged; Aged, 80 and over; Animals; Apoptosis; Calcimycin; Calcium; Calcium-Binding Proteins; Calpain; Caseins; Cataract; Eye Proteins; Humans; Immunoblotting; In Situ Nick-End Labeling; Ionophores; Lens, Crystalline; Macaca mulatta; Models, Animal; Reverse Transcriptase Polymerase Chain Reaction; Staining and Labeling

As a model of the cell proliferation occurring in posterior capsule opacification (PCO), lens epithelial cells (LEC) from human and rabbit capsulotomies, and a rabbit LEC line (N/N1003A) were grown in Dulbecco's Minimal Essential Media (MEM) with 10% fetal calf serum. LEC were exposed to the calcium ionophore, calcimycin, and viability was assessed by trypan blue staining, growth by 3H-thymidine incorporation and apoptosis by annexin/propidium iodide staining, calcein AM/ethidium bromide staining and DNA laddering. Human capsulotomy samples were similarly exposed to calcimycin, and apoptosis assayed by calcein AM/ethidium bromide staining. Calcimycin exposure induced apoptosis in both rabbit LEC cultures and human LEC, and changes leading to apoptosis could be detected within 30 minutes of calcimycin treatment. The decrease in viability and growth in human and rabbit LEC was dose-dependent. These data support the further evaluation of apoptosis induction as a possible treatment mechanism to prevent development of PCO following primary cataract surgery in humans.

Topics: Animals; Apoptosis; Calcimycin; Cataract; Cell Line; Cell Survival; Epithelial Cells; Female; Humans; Ionophores; Lens, Crystalline; Male; Rabbits

The purpose of the present experiments was to provide a biochemical mechanism for the involvement of lens-specific calpain Lp82 in experimental cataractogenesis in mice.. Nuclear cataracts were produced by culturing lenses from 4-week-old mice and rats in calcium ionophore A23187 or by injection of buthionine sulfoximine (BSO) into 7-day-old mice. Casein zymography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblot analysis, calcium determinations, in vitro precipitation, and cleavage site analysis by mass spectrometry were performed on lens samples.. Amino acid sequences for Lp82 were found to be highly conserved in lenses from mouse to cow, and expressed Lp82 proteolytic activity was high in the mouse and rat. Lenses from mice were more susceptible to A23187-induced cataract and BSO cataracts than rats. Both types of cataracts showed rapid elevation of calcium, activation of Lp82 and m-calpain, and proteolysis of crystallins. Lp82 caused in vitro precipitation of crystallins; and in contrast to m-calpain, Lp82 truncated only the first five amino acids from the C-terminus of alphaA-crystallin.. Under pathologic conditions of massive elevation of lens calcium found in young rodent lenses, overactivation of Lp82 and m-calpain leads to rapid truncation of crystallins at both common and unique cleavage sites, precipitation of truncated crystallins, and cataract.

Topics: Amino Acid Sequence; Animals; Buthionine Sulfoximine; Calcimycin; Calcium; Calpain; Cataract; Cysteine Endopeptidases; Electrophoresis, Polyacrylamide Gel; Immunoblotting; Lens, Crystalline; Light; Mass Spectrometry; Mice; Mice, Inbred ICR; Molecular Sequence Data; Organ Culture Techniques; Rats; Rats, Sprague-Dawley; Scattering, Radiation; Spectrophotometry, Atomic

To compare effects of calpain inhibitors on in vitro light-scattering in rat lens soluble protein and calcium-ionophore (A23187)-induced cataract formation in cultured rat lenses.. Rat lens soluble protein was hydrolyzed for 24 hours by activation of endogenous lens calpain. Ten calpain inhibitors were tested in this model at 10 and 25 microM concentration. As an index of protein precipitation, light scattering was measured daily at 405 nm for 8 days. Lens proteins were analyzed by isoelectric-focussing. Subsequently, rat lenses were cultured for 5 days with 10 microM A23187. Calpain inhibitors (SJA6017, MDL28170, AK295 and PD150606), which inhibited light-scattering were tested at 100 microM concentration in this model. Cataract evaluation, isoelectric-focussing and calcium determinations were performed.. At 25 microM concentration AK295, SJA6017, E-64, PD-150606 and MDL28170 produced greater than 25% inhibition of light-scattering. Isoelectric-focussing revealed that addition of Ca(2+) produced characteristic crystallin proteolysis and aggregation patterns. AK295, SJA6017, MDL28170 and E64c prevented these changes. Lenses cultured in A23187 exhibited nuclear cataract, elevated calcium and proteolysis and aggregation of crystallins. Co-culture with SJA6017, MDL28170 and E64c reduced A23187-induced nuclear opacities, proteolysis and aggregation of crystallins without affecting increased total calcium.. Endogenous calpain-activation model and A23187-induced cataract model can be used sequentially to screen calpain inhibitors for potential anti-cataract activity. Proteolytic changes in lens cortex after exposure to A23187 are also due to calpain activation. AK295, SJA6017 and MDL28170 possess efficacy against calcium-induced models of rodent cataracts. Use of calpain inhibitors represents a promising approach to cataract therapy.

Topics: Animals; Calcimycin; Calcium; Calpain; Cataract; Chemical Precipitation; Crystallins; Cysteine Proteinase Inhibitors; Isoelectric Focusing; Lens Nucleus, Crystalline; Light; Organ Culture Techniques; Rats; Rats, Wistar; Scattering, Radiation

To compare calcium ionophore-induced cataract formation and in vitro light scattering in cultured lenses from guinea pig and rabbit.. Lenses from guinea pig and rabbit were cultured for 5 or 6 days with calcium ionophore A23187. To assess the involvement of calpain in cataract formation; SDS-PAGE, immunoblotting and calcium determinations were performed. For in vitro light scattering, lens soluble proteins from rabbit were hydrolyzed for 24 h by either endogenous lens calpain, or by addition of purified m-calpain and then further incubated for up to 10 days. Light scattering was measured daily at 405 nm.. Lenses from younger guinea pigs cultured in A23187 first developed outer cortical opacities followed by nuclear cataract. Total calcium was markedly increased by A23187 in lenses of all ages. Proteolysis of crystallins and alpha-spectrin were observed in nuclear cataract in younger guinea pigs. This was attenuated with age, in association with the attenuation of cataract formation with age. Calpain 80 kDa subunit in the lenses cultured with A23187 was also decreased. Co-culture with SJA6017 or E64d (reversible and irreversible inhibitors of calpain, respectively) reduced A23187-induced nuclear opacities, proteolysis of crystallins and alpha-spectrin, and loss of calpain without affecting increased total calcium. In contrast, rabbit lenses cultured in A23187 did not develop nuclear cataract, although biochemical changes in cultured rabbit lenses were similar to those in cultured guinea pig lenses. Furthermore, no appreciable in vitro light scattering occurred in soluble proteins from rabbit lenses after activation of endogenous m-calpain, or after addition of exogenous purified m-calpain, although crystallins were partially hydrolyzed by calpain.. Both rabbit and guinea pig lenses undergo calpain-induced proteolysis upon elevation of lenticular calcium. However, factors in intact guinea pig lenses may promote light scattering and insolubilization after proteolysis by calpain, but these factors were not functional in rabbit lenses. Discovery of the factors promoting light scatter and insolubilization after proteolysis will help to explain the role of certain crystallin polypeptides in cataract formation.

Topics: Animals; Body Water; Calcimycin; Calcium; Calpain; Cataract; Crystallins; Electrophoresis, Polyacrylamide Gel; Guinea Pigs; Ionophores; Lens Nucleus, Crystalline; Light; Organ Culture Techniques; Organ Size; Rabbits; Scattering, Radiation; Spectrin

Previous studies have shown that calcimycin induces cataract in organ culture. To investigate the mechanism of this induction, the viability of lens epithelial cells in calcimycin (calcium ionophore, A23187)-treated rat lenses were examined. During incubation of lenses with 5 microM calcimycin, apoptotic epithelial cells were found after a 2-hr treatment as determined by terminal deoxynucleotidyl transferase (TdT) labeling. The percentage of apoptotic cells quickly rose as the incubation time increased. After a 12-hr incubation, more than 60% of the lens epithelial cells underwent apoptosis. Prolonged c-fos expression, previously shown to be an indicator of programmed cell death, was also observed during this treatment. DNA fragmentation assays further confirmed that the TdT labeled cells were indeed apoptotic. Under the same incubation conditions, the cultured lenses gradually lost transparency and became completely opaque in about 30 hr. Since the vertebrate lens contains only a single layer of epithelial cells, apoptotic death of these cells activated by calcimycin quickly destroys the lens epithelium, impairs homeostasis of the underlying fiber cells and initiates development of lens opacification.

Topics: Animals; Apoptosis; Calcimycin; Cataract; Cells, Cultured; Chromatin; DNA; Epithelium; Genes, fos; Lens, Crystalline; Rats; Rats, Sprague-Dawley

To determine if the susceptibility of rat lenses to cataract formation in culture changes with increasing age and to investigate the regional differences in crystallin degradation and insolubilization during the formation of cataracts in cultured lenses.. Lenses from 4-week-old (young group) and 12-week-old (adult group) rats were divided into four subgroups: noncultured control, cultured control, cultured in calcium ionophore A23187, and cultured in ionophore plus calpain inhibitor E64. Lenses were cultured for 7 days, and the cortex and nucleus were homogenized and separated into water-soluble and water-insoluble fractions. Two-dimensional electrophoresis and N-terminal sequencing were then performed.. Young lenses treated with ionophore produced thin cortical and dense nuclear opacities. Adult lenses treated with ionophore also developed thin cortical opacity, but no nuclear opacity was observed, even though a large increase in the concentration of insoluble protein occurred. Two-dimensional electrophoresis and sequencing suggested that calpain caused protein degradation in the cortex region. However, unlike nuclear opacity, the formation of opacity in the cortex was not inhibited by E64 in young or adult lenses.. Calpain was activated, and crystallins were proteolyzed in the cortex of ionophore-treated lenses. However, cortical opacity was not the result of proteolysis by calpain. Maturation also decreased the susceptibility of rat lens nucleus to calcium ionophore cataract.

Topics: Aging; Amino Acid Sequence; Animals; Calcimycin; Calpain; Cataract; Crystallins; Cysteine Proteinase Inhibitors; Lens, Crystalline; Leucine; Molecular Sequence Data; Organ Culture Techniques; Protein Denaturation; Rats; Rats, Sprague-Dawley; Solubility

To determine if limited proteolysis of beta-crystallins is associated with insolubilization of proteins in rats lens during maturation and to test if the protease, calpain II, is involved.. Soluble and insoluble lens proteins from 4-day-old to 4-month-old rat lens cortexes and nuclei were separated by two-dimensional electrophoresis. The insoluble proteins from 4-month-old nuclei were electroblotted and the NH2 termini of proteins sequenced. Cleavage sites appearing at 4 months of age were compared to cleavage sites produced by purified calpain II and to cleavage sites appearing in cataracts induced by selenite in vivo or in lenses cultured with calcium ionophore A23187 or diamide.. In solubilization of more than 50% of proteins occurred in the nucleus of the transparent rat lens by 4 months of age. The insoluble protein that formed contained an abundance of partially degraded beta-crystallin polypeptides missing portions of their NH2 terminal extensions. In contrast, these truncated beta-crystallins were largely absent from both the cortex and soluble fraction of the nucleus. The cleavage sites in the insoluble beta-crystallins appearing during maturation in the lens nucleus were similar to cleavage sites produced by purified calpain II and also similar to cleavage sites appearing in the insoluble protein of cataractous lenses.. These results suggest that proteolysis of beta-crystallins by the protease calpain II contributes to protein insolubilization during lens maturation and that acceleration of this insolubilization process is associated with cataract formation in rodent lenses.

Topics: Amino Acid Sequence; Animals; Calcimycin; Calpain; Cataract; Crystallins; Diamide; Electrophoresis, Gel, Two-Dimensional; Lens, Crystalline; Molecular Sequence Data; Organ Culture Techniques; Peptide Fragments; Peptide Mapping; Rats; Rats, Sprague-Dawley; Sodium Selenite; Solubility

E64, an inhibitor of calpain (EC 3.4.22.17) and other cysteine proteases, slows the rate of formation of cataract in cultured rat lenses. The purpose of this study was to determine (1) why E64, a charged compound with little cell permeability, was effective in reducing cataract in cultured lens and (2) whether uncharged more permeable protease inhibitors are more effective than E64 in preventing cataract. Results showed that E64 entered the lens, but only after the lens was treated with the calcium ionophore, A23187, or sodium selenite, both of which cause cataracts. Therefore, the uptake and subsequent effectiveness of E64 may be related to a generalized increase in membrane permeability during induction of cataract in culture. Three protease inhibitors, reported to have improved cell permeability, were compared with E64 for their ability to prevent cataracts in cultured lenses. cBz-ValPheH, calpain inhibitors I and II, are uncharged-aldehyde inhibitors of calpain. Calpain inhibitors I and II even at high concentrations were not effective at reducing lens opacity caused by calcium ionophore and were toxic to the lens. cBz-ValPheH, which is slightly toxic to the lens, was able to significantly reduce lens opacity induced by calcium ionophore. The presented data suggest that while E64 decreases cataract formation in cultured lens, the more cell permeable inhibitor, cBz-ValPheH, may have greater efficacy as an anticataract drug in vivo.

Topics: Amino Acid Sequence; Animals; Calcimycin; Calpain; Cataract; Cell Membrane Permeability; Chromatography, High Pressure Liquid; Culture Techniques; Cysteine Proteinase Inhibitors; Dipeptides; Drug Interactions; Electrophoresis, Polyacrylamide Gel; Glycoproteins; Lens, Crystalline; Leucine; Leupeptins; Molecular Sequence Data; Rats; Rats, Sprague-Dawley

The purposes of this experiment were: (1), to compare effect of three E64 derivatives, E64, E64c and E64d in preventing nuclear opacity and proteolysis in calcium ionophore-induced cataract and (2), to measure the accumulation of E64 derivatives in the cultured lenses. In vitro E64 and E64c strongly inhibited purified calpain II from porcine heart, while E64d showed weaker inhibition than E64 and E64c. In cultured lenses, all three E64 derivatives reduced nuclear opacity by calcium ionophore A23187 in a concentration-dependent manner, and E64d, the ethyl-ester of E64c, was the most effective. When lenses were cultured in E64d for 2 h, the resulting concentration of E64 derivative in the lens was markedly higher than during culture in E64 or E64c. All three E64 derivatives prevented proteolysis of crystallins seen in A23187 cataract. The stronger effect of E64d against A23187 cataract was likely due to an earlier penetration into the lens, conversion to E64c and inhibition of activated calpain.

Topics: Animals; Calcimycin; Calcium; Calpain; Cataract; Crystallins; Cysteine Proteinase Inhibitors; Lens, Crystalline; Leucine; Organ Culture Techniques; Organ Size; Rats; Rats, Sprague-Dawley

Cataracts were produced in cultured rat lenses by either 10 microM calcium ionophore A23187, 25 microM sodium selenite, or 30 mM xylose. E64, an inhibitor of cysteine proteases, such as calpain (EC, 3.4.22.17), reduced severity of cataract and proteolysis of crystallins when included at a 500 microM concentration in the culture medium along with cataractogenic agents. Calpain II enzyme activity and the amount of calpain antigen were decreased in the cytosol of cataractous lens. However, E64 caused an increase in the amount of an 80-kD calpain subunit associated with the ethyleneglycol-bis-(beta-aminoethylether) tetraacetic acid/ethylenediaminetetraacetic acid-washed insoluble proteins when lenses were incubated with cataractous agents. These data indicate that E64 was at least partially effective in inhibiting lens calpain, and that activation of lens calpain may involve binding to the insoluble fraction. These results provide strong evidence for the activation of calpain in rodent cataracts and suggest testing inhibitors of calpain as anticataract drugs.

Topics: Animals; Calcimycin; Calpain; Cataract; Chromatography, High Pressure Liquid; Crystallins; Culture Techniques; Cysteine Proteinase Inhibitors; Electrophoresis, Polyacrylamide Gel; Immunoblotting; Lens, Crystalline; Leucine; Rats; Rats, Inbred Strains; Selenium; Sodium Selenite; Xylose